Deletion of the leader peptide of the mitochondrially encoded precursor of Saccharomyces cerevisiae cytochrome c oxidase subunit II.

Cytochrome c oxidase subunit II (Cox2p) of Saccharomyces cerevisiae is synthesized within mitochondria as a precursor, pre-Cox2p. The 15-amino acid leader peptide is processed after export to the intermembrane space. Leader peptides are relatively unusual in mitochondrially coded proteins: indeed mammalian Cox2p lacks a leader peptide. We generated two deletions in the S. cerevisiae COX2 gene, removing either the leader peptide (cox2-20) or the leader peptide and processing site (cox2-21) without altering either the promoter or the mRNA-specific translational activation site. When inserted into mtDNA, both deletions substantially reduced the steady-state levels of Cox2p and caused a tight nonrespiratory phenotype. A respiring pseudorevertant of the cox2-20 mutant was heteroplasmic for the original mutant mtDNA and a p- mtDNA whose deletion fused the first 251 codons of the mitochondrial gene encoding cytochrome b to the cox2-20 sequence. The resulting fusion protein was processed to yield functional Cox2p. Thus, the presence of amino-terminal cytochrome b sequence bypassed the need for the pre-Cox2p leader peptide. We propose that the pre-Cox2p leader peptide contains a targeting signal necessary for membrane insertion, without which it remains in the matrix and is rapidly degraded.

Isolation and characterization of a yeast strain carrying a mutation in the mitochondrial promoter for COX2.

Subunit II of cytochrome c oxidase is a mitochondrially encoded protein required for cellular respiration. A respiration-deficient yeast strain has been isolated and shown genetically to carry a mutation in the subunit II structural gene COX2. The respiration-deficient strain produces no subunit II polypeptide and is missing two major transcripts of the subunit II gene. The mutation, first mapped by rho- recombinational rescue to the 5' end of the gene, has been localized precisely, by DNA sequencing, 58 nucleotides upstream of the COX2 ATG initiation codon. The mutant strain carries a single nucleotide change (A to T) relative to the wild type sequence. This mutation occurs in a sequence with substantial homology to a previously identified yeast mitochondrial promoter consensus sequence. These results provide the first in vivo evidence for the importance of the consensus sequence for promoter function.

Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae.

We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies. The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration. Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants. Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes. A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains. We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.